Screening Of Thermostable Protease-producing Strains And Cloning,Expression Of Protease Gene

Collected the soil samples from turpan of Xinjiang province, and isolated176strains from the samples. One thermophilic bacterial strain with thermophilic protease activity using the study of function, transparent based on morphological characteristics and16S rRNA gene sequence analysis, The strain was identified as Bacillus tequilensis, named C9. The study of enzymatic properties shows that:The strains C9produce protease’s optimum reaction temperature was55℃, In80℃after30min treatment, the enzyme activity retained1%, the optimum pH was9.0, the thermal stability of the enzyme and pH stability, In the different metal ions after the treatment, Fe2+activate. The strain was shaking flask fermentation optimization of fermentation conditions, results show that, The main influence factors of the strain were glucose, liquid loading and inoculation. The main effect factors in PB were obtained after the test, the enzyme producing conditions were optimized by response surface analysis, analysis of variance and regression analysis on the experimental results, the two regression equation model, the optimal fermentation condition was:glucose1.3%, the volume of liquid45ml,5%inoculation quantity on this condition, the enzyme activity was86.17U/ml, compared with the non optimized enzyme activity increased2.1times.Protease gene was amplified by PCR and bioinformatics software for sequence analysis, construction of pET-28a-apr prokaryotic expression recombinant plasmid, the recombinant protein was expressed in E. coli BL21though IPTG in37℃and enzyme activity was determined. The gene fragment of protease gene apr was1098bp, the nucleotides sequence homology was98%, compared with Bacillus subtilis apr E (AB734697), encoded a mature protein containing299amino acids, which was easy to protein solution, strong hydrophilicity, the protein belonged to the peptidases_S8_S53superfamily protein family, the molecular mass of fusion protein was29kDa, protease activity was28.4u/mL. Application of inclusion body protein purification kit for purification of the protein, and kinetic studies showed that, the protein purity was95%, The substrate was casein with pH7.2, temperature45℃, Bacillus(Bacillus tequilensis C9) protease Vmax value is130.12umol/L.min, Km value is0.655g/L.Relying solely on the natural separation of microorganisms breeding, due to low mutation rate, mutation rate is small, it is difficult to obtain high yield strains by induced mutation, which is the most reliable method for microbial breeding. This study adopts ultraviolet and NTG composite mutagenesis breeding, a high-yielding strain N-12, compared with the original strain, the enzyme activity increased12.68%.